This invention pertains to methods and devices for the detection and/or enumeration of microorganisms. More particularly this invention pertains to methods and devices for the detection and/or enumeration of microorganisms in opaque and/or highly pigmented products.
The presence and enumeration of microorganisms in industrial samples (food, beverage, dietary supplements, cosmetics, toiletry, etc.) have been traditionally determined by growing the microorganisms in agar in Petri dishes and counting the colonies. In the last two decades, other tests have been practiced for industrial samples. These methods are based on culturing the sample in liquid media and monitoring the metabolites generated during the growth of the microorganisms. Several systems, such as the Bactometer (bioMerieux, Hazelwood, Mo. USA), BacTrak (Sy-Lab, Neupurkersdort, Austria), Malthus Systems (Lab M, Crawley, UK) and the RABIT (Bioscience International, Bethesda, Md. USA), are based on monitoring the electrical properties of the growth media measured via two metallic electrodes immersed in the liquid media. The conductance and capacitance of the electrode-media combination is measured by imposing AC electrical current via the electrodes in the media.
Another practical approach has been developed by the present inventor is described and claimed in U.S. Pat. No. 5,366,873. This approach is particularly suitable for assessing food, dairy, and beverage samples. With this approach, the test container contains two distinct phases: (a) a liquid phase formed from a mixture of growth media and an indicator substrate, and (b) a semi liquid phase, comprising a semi-liquid layer, such as agar, and identical liquid compounds present in the liquid phase. Optical readings are periodically performed to detect the optical transmittance of the semi liquid phase utilizing a light source and a photo detector placed on the opposite sides of the layer. Liquid molecules and ions can quickly diffuse between the two phases which are in equilibrium. The diffusion rate is high, and its consistency makes it adequate for enumeration tests utilizing the following equation:Log(CFU)=A−B×tD Wherein CPU is the Colony Forming Units (i.e. the number of microorganisms in the sample). A and B are constants, and tD is the Detection Time which is the point in time (hours) in which the concentration of the microorganism in the liquid media exceeds a specific threshold (around 106 cells/ml). At this point optical readings through the semi liquid phase start following the exponential growth pattern of the microorganisms.
This device is not without some drawbacks. It cannot measure highly pigmented samples such as colored beverages, gelatin capsules for pharmaceutical and veterinary products, dyes, or blood. During the incubation period the pigments can diffuse into the semi liquid phase and mask the optical readings. Another disadvantage of this device is that the semi-liquid phase (agar) disintegrates in higher temperatures and therefore, the device cannot be thermally sterilized. Consequently, it cannot be used reliably for clinical and sterility tests. Another disadvantage of this device is that the agar occasionally gets dislodged during shipping, particularly when exposed to low and freezing temperatures.
Another practical approach of culturing and monitoring microorganisms (bacteria, yeasts and molds) in test samples in the presence of interfering materials has been developed and successfully commercialized utilizing opaque silicone based optical indicator matrices. One such product has been described by Turner, et al. (U.S. Pat. No. 4,945,060), Calandra, et al. (U.S. Pat. No. 5,094,955), Thorpe, et al. (U.S. Pat. No. 5,162,229), Di Guiseppi, et al. (U.S. Pat. No. 5,164,796), and Turner, et al. (U.S. Pat. No. 5,217,876). The basic principle of this device is to affix a disposable sensor to the interior surface of a transparent container that can monitor the production of CO2 when the microorganisms grow and metabolize. The sensor comprises a solid composition with an optical indicator substrate immobilized within it which is placed Hush against the inside surface of the transparent container, such that the indicator substrate is visible from outside. In this device the sensor is separated from the specimen and its growth media by a solid layer that permits the passage of gas molecules but prevents passage of liquid and particulate matter.
These devices are therefore characterized by two distinctive phases: (a) liquid phase that includes the growth media where the specimen or sample is incubated and (b) solid phase in which the indicator substrate is embedded. Unlike the previous device described in U.S. Pat. No. 5,366,873, no growth media is present in the solid phase and no indicator substrate is present in the media. The solid sensor completely eliminates penetration of liquid and interfering substances including pigmentation molecules, thereby enabling monitoring of highly colored samples.
These devices are slow in reacting to the generation of CO2 by the metabolizing microorganisms. First, the chemical composition of the sensor makes it opaque to light, requiring an instrument that measures optical reflectance. Second, the sensor is placed at the bottom of the container, such that one of its flat surface interfaces with the liquid media, while the other surface faces the transparent bottom. The generated CO2 gas has to diffuse along the vertical axis of the sensor until it can vary the optical characteristics of its bottom surface. Since the diffusion rate of the gas in the solid matrix is slow (several hours), the detection of the microorganisms can be severely delayed, which can be critical to patient's life. In addition, the manufacturing process of affixing the sensor to the container and the nature of reflectance optics, result in inherent variability of the optical measurements as related to each device's sensor. Consequently, this sensor is inadequate for enumeration tests and is limited only to presence/absence determinations.